Characterisation of the fungal ligands, structure and function of the C-type lectin receptor, MelLec

真菌感染的发病率在过去30年中显著增加，主要是因为艾滋病毒/艾滋病的流行和侵入性医疗干预。主要由皮肤真菌引起的浅表真菌病影响约25%的世界人口，而侵袭性真菌感染导致超过50%的死亡率。迫切需要充分了解我们的免疫系统用于对抗真菌感染的机制，因为这将有助于开发诊断工具和新的治疗方法。针对真菌病原体的保护最初由先天免疫系统通过模式识别受体（PRR）识别保守的微生物相关分子模式（PAMP）来提供。对于抗真菌免疫至关重要的PRR家族之一是C型凝集素受体（CLR）。我们已经确定了一种新的受体，黑色素传感C型凝集素受体（MelLec），它在抗真菌免疫中起着重要作用，因为它识别烟曲霉孢子中的1，8-二羟基萘（DHN）-黑色素。与抗真菌免疫中的其他必需CLR不同，鼠MelLec在非髓样细胞中表达，并且由CD 31+内皮细胞普遍表达。有趣的是，鼠MelLec也由CD 31+细胞亚群表达，该亚群异常地共表达上皮标志物EpCAM（CD 31 +EpCAM+）。这些双阳性细胞仅在小鼠肺和肝脏中检测到，其功能尚不清楚。使用MelLec敲除小鼠，证明该受体是保护免受A.在烟曲霉和人类中，该受体的单核苷酸多态性对炎症反应产生负面影响，并显著增加干细胞移植受体对播散性曲霉感染的易感性。在这个项目中，我们将进一步探索这种MelLec在抗真菌免疫中的作用，并确定除了黑色素之外的配体是否被识别。DHN-黑色素的生物合成途径在A.烟曲霉中，并通过筛选突变体，我们确定MelLec的配体七肽萘并吡喃酮（YWA 1）是在途径的第一步期间由聚酮酶合成的。我们已经证明，纯化形式的YWA 1可以抑制可溶性融合蛋白Fc-MelLec与A.烟曲霉分生孢子聚酮酶参与许多其他真菌次级代谢产物的产生，在本项目中，在流式细胞术测定中使用我们的Fc-MelLec，我们已经表明许多真菌物种的次级代谢产物（例如A.烟曲霉（Penicillium fumigatus）、青霉属（Penicillium species））是该受体的配体。将使用适当的转染和原代人和小鼠细胞系在体外阐明MelLec识别真菌次级代谢产物后诱导的细胞应答。待检查的应答包括例如MelLec介导的次级代谢物的摄取（通过流式细胞术和基于ELISA的方法）和细胞因子和趋化因子的产生。鉴于真菌毒素对人类疾病的巨大影响，我们将探讨MelLec在真菌次级代谢产物致癌活性中的作用，使用已建立的体内肝癌模型。在本项目中，我们还将培养表达MelLec的CD 31 +EpCAM+细胞。为此，我们将从小鼠肺中分离出CD 31 +EpCAM+细胞，然后进行单细胞分选和单细胞RNA测序。由于这些细胞表达MelLec，其识别A.通过对烟曲霉的研究，我们可能会发现它们在抗真菌免疫或一般免疫中起着重要作用。此外，由于MelLec在CD 31 +EpCAM+细胞中的表达是组织特异性的，这些细胞可能对肺具有独特的功能，这可能是目前未知的。总之，该项目将大大提高我们对MelLec在免疫和疾病中的作用和功能的认识。

The incidence of fungal infections has increased significantly over the last 30 years mainly because of the HIV/AIDS pandemic and invasive medical interventions. Superficial mycoses caused mainly by dermatophytes affect approximately 25% of world population while invasive fungal infections lead to more than 50% mortality. There is an urgent need to fully understand the mechanisms that our immune system employs to fight fungal infections as this will aid in the development of diagnostic tools and novel treatments. Protection against fungal pathogens is initially provided by the innate immune system via the recognition of the conserved microbial-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). One of the families of PRRs that are critical for antifungal immunity are the C-type Lectin Receptors (CLRs). We have identified a novel CLR, the Melanin sensing C-type Lectin receptor (MelLec), that plays an important role in antifungal immunity as it recognises 1,8-dihydroxynaphthalene (DHN)-melanin in conidial spores of Aspergillus fumigatus. Unlike the other essential CLRs in antifungal immunity, murine MelLec is expressed in non-myeloid cells and is ubiquitously expressed by CD31+ endothelial cells. Interestingly, murine MelLec is also expressed by a sub-population of CD31+ cells that unusually co-express the epithelial marker, EpCAM (CD31+EpCAM+). These double-positive cells are only detected in the mouse lung and liver and their function is unknown. Using MelLec knock out mice this receptor was demonstrated to be required for protection against disseminated infection with A. fumigatus and in humans, a single nucleotide polymorphism of this receptor negatively affects the inflammatory responses and significantly increases susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. In this project, we will further explore the role of this MelLec in antifungal immunity and determine if ligands in addition to melanin are recognised. The biosynthetic pathway of DHN-melanin is well characterised in A. fumigatus and by screening mutants we determined that the ligand heptaketide napthopyrone (YWA1) of MelLec was synthesised by polyketide synthases during the very first step of the pathway. We have shown that a purified form of YWA1 can inhibit a soluble fusion protein Fc-MelLec from binding to A. fumigatus conidia. Polyketide synthases are involved in the generation of many other fungal secondary metabolites, and in this project, using our Fc-MelLec in flow cytometry assays, we have shown that secondary metabolites of many fungal species (e.g. A. fumigatus, Penicillium species) are ligands for this receptor. Cellular responses induced by MelLec following recognition of fungal secondary metabolites will be elucidated in vitro using appropriate transfected and primary human and mouse cell lines. Responses to be examined include, for example, MelLec-mediated uptake of secondary metabolites (by flow cytometry and ELISA based methods) and the production of cytokines and chemokines. Given the huge impact of mycotoxins on human disease, we shall explore the role of MelLec in the carcinogenic activities of fungal secondary metabolites, using established in vivo models of hepatocarcinogenesis. In this project, we will also characterise the CD31+EpCAM+ cells that express MelLec. To do this, we will isolate the CD31+EpCAM+ cells from mouse lungs and then we will perform single-cell sorting and single-cell RNA sequencing. Since these cells express MelLec, which recognises 1, 8-DHN melanin of A. fumigatus, we might find that they play a significant role in anti-fungal immunity or immunity in general. Also, because the expression of MelLec in CD31+EpCAM+ cells is tissue specific, these cells might have a distinct function on the lung which might be currently unknown. In sum, this project will significantly advance our knowledge of the role and function of MelLec in immunity and disease.